PROJECT SUMMARY/ABSTRACT Cardiovascular diseases (CVD) are the leading cause of death in the US and other developed nations. In 2009 alone, more than 80 million Americans suffered from CVD with associated cost $313 billion. The development of CVD and other chronic circulatory disorders has been associated with exposure to drugs and environmental toxicants. These chemicals can alter the bioavailability of a key regulator of vascular function ? nitric oxide (NO), which is produced by endothelial cells (ECs). The impairment of vascular EC NO production can contribute to the development of chronic circulatory disorders. However, this does not explain why some individuals develop the disease and others do not. Accordingly, the National Institute of Environmental Health Sciences leads in efforts to understand the contribution of individual susceptibility to complex diseases, such as CVD. To aid these efforts, Creative Scientist, Inc. (CSI) is developing advanced tools enabling the generation of data on individual vascular susceptibility to drugs and environmental toxicants. Using proprietary protocols and cell growth media supplements, CSI isolates Endothelial Colony-Forming Cells (ECFC) from small volumes of peripheral blood and cryopreserves the cells. ECFCs are unique primary cells involved in both embryonic and adult vasculogenesis. The donor-specific nature and robust growth potential of ECFCs allow for the testing of tens of thousands of chemicals in cells derived from any number of individuals. Our goal is to commercialize a NO testing service using a novel and highly specific NO reagent and a high content imaging platform. In Aim 1, we will determine the sensitivity and selectivity of our novel NO-specific fluorescent probe. Completion of this Aim will demonstrate the utility of our reagent for measuring basal and induced NO levels in ECFCs. In Aim 2, we will determine the reproducibility of measuring NO levels in different ECFC lineages using different culture conditions (e.g., with inducer, with inducer and inhibitor). Completion of Aim 2 will establish the utility of ECFCs for identification of NO inhibitors. In Phase II, we will test the assay's ability to detect human response variability to 1,000 toxicants using a collection of ECFCs derived from up to 500 donors. Our ultimate goal is to provide a commercial service to regulatory and research agencies and pharmaceutical companies aiming to evaluate the potential vascular toxicity of environmental toxicants and drug candidates in a population and, in particular, trying to identify both sensitive and resistant individuals and/or population sub-groups. The success of our assay development efforts will help subsequently commercialize a stand-alone NO testing kit.